Signaling and CD4 T cell activation is associated with the formation of an immunological synapse. There is considerable information describing signaling pathways, but little is know about the series of molecular interactions that lead to the formation of an immunological synapse and about the significance of synapse formation to T cell activation. The formation of a synapse depends on binding of the T cell receptor to peptide/MHC complexes expressed on antigen presenting cells. Following initial T cell receptor signaling, the complexes of T cell receptor bound to peptide/MHC migrate to the interface between the T cell and antigen presenting cell, with eventual segregation of molecules into a central core of T cell receptors and peripheral ring of CD4 and LFA-1/ICAM pairs. How T cell receptor ligation leads to these series of events is still largely unresolved. Dr. Bottomly's laboratory has observed that subsets of CD4 T cell (naive, Th1, Th2) differ in immunological synapse formation. The focus of this proposal is to study the mechanism by which T cell receptor clustering and immunological synapse formation is regulated. In Aim l, how the spacial organization and signaling function of CD4 influences: 1) T cell receptor clustering at the T/antigen presenting cell interface; 2) formation of early microclusters of TCR at the synapse site; 3) physical association of CD4 with the engaged T cell receptor; and 4) differences in T cell receptor clustering observed in Thl and Th2 cells will be determined. In aim 2, differences in the regulation of mature immunological synapse formation in subsets of mature CD4 T cell by analyzing: 1) early and late events in synapse formation in naive, Th1, Th2, and memory cells; 2) the role of CD28/CTLA-4 signaling in clustering and synapse formation in CD4 subsets; 3) in vivo synapses of subsets of CD4 T cells will be determined. It is anticipated that understanding how the T cell receptor signaling complex is compartmentalized will provide a greater insight to unique signaling mechanism used by subsets of CD4 T cell.